The objective of the present proposal is to gain basic information on the sequence and mechanism of the respiratory chain linked energy transduction and the structure-function relationship of mitochondrial inner membrane by studying various types of the respiratory chain-linked electron and energy transfer reactions in a bovine cardiac submitochondrial system. The following 3 approaches will be employed: (1) Purification and characterization of homogeneous membrane preparations by means of density gradient centrifugation and biophysical and biochemical analyses. (2) Investigation of the functional aspects of the mitochondrial inner membrane: A. Localization of NADH dehydrogenase and cytochromes b and c1 in the mitochondrial inner membrane by means of resolution and reconstitution. B. Ion translocation in submitochondrial membrane. Special attention will be directed to the relationship between the kinetics of cation translocation and electron transfer or other energy linked functions. (3) Application of extrinsic probes: for the study of membrane conformation and/or state changes. Continued effort on the environmental sensitive probes, 8-anilino-naphthalene-1-sulfonate (hydrophobicity) and quinacrine (pH) will be made. Derivatives of these probes are planned to be made and investigated. Modification of submitochondrial membrane is also planned in order to elucidate the mechanism of the fluorescence changes associated with the functional activities of the membrane. This type of attack should expose the inadequacies of the existing hypotheses and theories, lead to new theories, and clarify the delicate organization of the mitochondrial energy transfer system and ultimately lead to the understanding of the regulation of the mitochondrial energy metabolism in both healthy and diseased cardiac muscle.